Solution containing carnitine for the storage and perfusion of organs awaiting transplantation

ABSTRACT

A storage solution to maintain and perfuse organs awaiting transplantation comprising (a) an isotonic balanced solution comprising a physiologically acceptable amount of potassium, mono acidic phosphate, biacidic phosphate, chloride, sodium and bicarbonate ions; (b) 50-250 mM glucose; (c) 0.2-20 mM of an alkanoyl L-carnitine or a physiologically acceptable salt thereof; (d) 1-100 mM of L-carnitine or a physiologically acceptable salt thereof; (e) water is described. The storage solution can also include other components such as anti-oxidants and/or chelating agents.

This application is a continuation of U.S. application Ser. No.10/480,824, now U.S. Pat. No. 7,422,844, which was filed on Dec. 15,2003, which is the U.S. national stage of PCT/IT02/00391, filed on 13Jun. 2002, which claims priority to and the benefit of ItalianApplication No. RM2001A000337 filed on 14 Jun. 2001, the contents ofwhich are incorporated herein by reference in their entirety.

The present invention refers to a solution for the storage and perfusionof organs awaiting transplantation.

BACKGROUND OF THE INVENTION

In the last 10 years, orthotopic organ transplant has become anirreplaceable therapeutic method for patients having particular,terminal stage organ diseases, such as for example hepatic, cardiac,pancreatic, pulmonary and renal diseases.

However, rejection of the transplanted organ continues to be asubstantial problem.

For example, rejection contributes to a mortality rate of 15-25% duringthe first year after surgery in the case of liver transplantation(Strasberg S. M. et al., Hepatology 1994, 20: 829).

The phases in which the liver for transplantation undergo damage havebeen determined as:

-   1) Heat ischemia during the removal from the donor;-   2) Cold ischemia during the hypothermic storage phase;-   3) Reperfusion of the organ in the recipient; (Transplantation    53:957-978, 1992).

The basic strategy for the storage of organs for transplantation is thatof slowing down the cellular catabolic processes through lowering thetemperature of the organ from 37° C. to around 4-6° C. (hypothermia).

Hypothermia lowers the metabolic rate and the rate of hydrolysiscatalysed by various intracellular enzymes, but does not completelyinhibit cellular metabolism. This can bring about processes which leadto cellular alterations both in the endothelial sinusoidal compartmentand in the hepatocytic compartment.

In fact, cooling of the isolated liver without perfusion results in arapid reduction in ATP and ADP levels (J.Surg.Res. 23:339-347, 1977;Cryobiology 31:441-452, 1994) in as much as the residual energy demandsexceed the cellular capacity to generate ATP through anaerobicglycolysis from glycogen reserves, with the consequent accumulation oflactic acid and intracellular acidosis.

The degradation of ATP to ADP and successively to AMP and adeninecauses, during hypothermia, an accumulation of hypoxanthine with theconcomitant conversion of xanthine dehydrogenase to xanthine oxidase,and is associated with increases in intracellular calcium and proteaseactivation (McCord J. M., N. Engl. J. Med. 1985, 312: 158).

In the reperfusion state, this results in degradation of hypoxanthine toxanthine and then to uric acid with the production of reactive oxygenintermediates (ROI) and oxidative type damage.

Furthermore, the diminished activity of enzymes, such as, for example,the Na/K ATPases results in changes in the conditions of electrolytebalance with consequent water influx and cellular swelling.

In recent years, a direct correlation between ATP content duringhypothermic storage of the organ for transplantation, and the success ofthe transplant in humans has been demonstrated (Hepatology 1988, 8:471).

To overcome such disadvantages, storage solutions whose composition hasbeen studied to counteract the dangerous effects of anoxic hypothermia(during the storage phase) and of the normothermic reperfusion (duringre-implantation phase) are used.

The formulation of such solutions is in continuous evolution to allowthe improvement of the vital state of the organ and the increase ofpreservation times.

Solutions useful for the storage of organs awaiting transplantation arealready known.

In Transplantation 2000 Apr. 15; 69(7):1261-5) it is reported that thesolution from the University of Wisconsin, known as UW solution, iscapable of retarding the catabolic processes and guaranteeing goodpreservation of the organs awaiting transplantation.

The composition of UW solution is based upon a pharmacological strategy,which intends to:

-   1) favour the re-synthesis of ATP through the addition of precursors    such as adenine and phosphate;-   2) prevent acidosis through the presence of phosphate buffer;-   3) inhibit xanthine oxidase activity through allopurinol;-   4) minimise the ionic redistribution using a composition similar to    that found intracellularly (high K⁺); and above all-   5) prevent cellular swelling through osmotic pressure, through the    addition of lactobionate, raffinose and high molecular weight    colloids, such as starch.

The basic strategy of this composition is however empirical, and it hasbeen hypothesised that the effect could derive from a phenomenon knownas “sum of protections” (Southard J. H. et al., Transplantation 1990,49: 251).

In Transplant Proc. 1999; August; 31(5):2069-70 it is reported that theCelsior solution is useful for the storage of organs awaitingtransplantation.

The saline solution EuroCollins is another known solution, useful forthe storage of organs, having the composition reported in the followingTable 1.

TABLE 1 Concentration K₂HPO₄•3H₂O 32 mM KH₂PO₄ 15 mM KCl 15 mM NaHCO₃ 10mM Glucose 194 mM 

This solution has been considered for many years the standard solutionin Europe for the storage of organs, in particular kidneys. Itsformulation is based on obtaining an electrolytic composition whichsimulates the intracellular environment. Further, in this solution,hypertonicity (420 mOsmol) is obtained by the addition of high glucoseconcentrations (around 190 mM).

The above-cited solutions are not without inconveniences, and have beensubject to numerous modifications.

The principal disadvantages shown by UW solution lie in its highviscosity, with consequent possible damage, above all sustained by theendothelial cells, during perfusion of the organ, and in the high costof each single component whilst remaining unsure as to theirindispensability [Transplant Proc. 1999; August; 31(5):2069-70.

Celsior solution presents the disadvantage of not being a suitablesolution for liver storage, if compared to solution UW [Transplant.2000; Oct. 27; 70(8):1140-2].

EuroCollins solution presents numerous disadvantages:

-   1—has a high glucose concentration which aggravates the problem of    acidosis, due to the enormous production of lactate during    hypothermic hypoxia;-   2—does not prevent cellular swelling during the storage of the    organ;-   3—is no better than solution UW (Transplantation 2000 Apr. 15;    69(7):1261-5).

The use of carnitines in the medical field is already known.

In Ann. Thorac. Surg. 2001; 71:254-9 the use of L-carnitine for thetreatment of cardioplegic ischemia, in isolated rabbit heart isdescribed. This work reports that L-carnitine shows a protective effecton the recovery of cardiac functions in isolated rabbit heart, when thiswas perfused with whole blood in to which L-carnitine had been added.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further described with reference to the appendeddrawings in which:

FIG. 1A is a diagram of a perfusion circuit, and

FIG. 1B is a diagram of an oxygenation and thermostatisation apparatus.

DESCRIPTION OF THE INVENTION

The use of carnitine in the preparation of a solution for the storage oforgans awaiting transplantation has never been previously described.

It has now been found that the addition of carnitine and/or an alkanoylL-carnitine to a storage solution for isolated organs exhibit asurprising capacity for storage and perfusion of organs, which issuperior to that of the known solutions previously mentioned.

The solution according to the present invention differs from the knownsolutions by the presence of L-carnitine and/or an alkanoyl L-carnitine.

The solution according to the present invention is suitable for use inthe storage and perfusion of organs awaiting transplantation.

Non-limiting examples of such organs are heart, liver, pancreas, lungand kidney.

Preferably, the solution according to the present invention comprisesL-carnitine and an alkanoyl L-carnitine, in which the alkanoylL-carnitine is selected from the group consisting of acetyl; propionyl;valeryl; isovaleryl; butiryl and isobutiryl L-carnitine.

In a general embodiment, the storage solution for maintaining andperfusing organs awaiting transplantation, according to the presentinvention comprises:

-   (a) a balanced isotonic solution comprising a physiologically    acceptable quantity of potassium ions, mono-acidic phosphate,    bi-acidic phosphate, chlorine, sodium, bicarbonate;-   (b) 50-250 mM glucose;-   (c) 0.2-20 mM alkanoyl L-carnitine or one of its physiologically    acceptable salts; and/or-   (d) 1-100 mM L-carnitine or one of its physiologically acceptable    salts;-   (e) water.    In a first preferred embodiment of the present invention the    solution for the preservation and perfusion of organs awaiting    transplantation (which will be hereinafter referred to as “Carnival    Solution”) has the composition reported in Table 2,

TABLE 2 Concentration K₂HPO₄•3H₂O  10-60 mM KH₂PO₄  3-50 mM KCl  3-50 mMNaHCO₃  2-50 mM Glucose 50-250 mM Alkanoyl L-carnitine or one of its 0.2-20 mM physiologically acceptable salts L-carnitine or one of itsphysiologically  1-100 mM acceptable salts

Isovaleryl L-carnitine is the preferred alkanoyl L-carnitine.

By physiologically acceptable salt of L-carnitine or of an alkanoylL-carnitine it is intended any salt thereof with an acid which does notgive rise to undesired toxic effects. These acids are well known topharmacologists and to experts in pharmaceutical technology.

Non-limiting examples of these salts are chloride, bromide, orotate,acid aspartate, acid citrate, magnesium citrate, acid phosphate,fumarate and acid fumarate, magnesium fumarate, lactate, maleate andacid maleate, mucate, acid oxalate, pamoate, acid pamoate, acidsulphate, glucosephosphate, tartrate, acid tartarate, magnesiumtartrate, 2-amino ethanesulphonate, magnesium 2-amino ethanesolphonate,choline tartrate and trichloroacetate.

Fumarate is preferred.

An example of a preferred solution, according to the present inventionis reported in table 3.

TABLE 3 Concentration K₂HPO₄•3H₂O 32 mM KH₂PO₄ 15 mM KCl 15 mM NaHCO₃ 10mM Glucose 187.5 mM   Isovaleryl L-carnitine Fumarate  2 mM L-carnitine(inner salt) 10 mM

The solution according to the present invention can contain in addition:

-   1) One or more antioxidants, in useful amount to prevent the    formation of free radicals derived from oxygen. Non-limiting    exasemples of such antioxidants are: allopurinol, glutathione,    beta-carotene, catalase, superoxide dismutase, dimethyl thiourea    (DMTU), diphenyl phenylene diamine (DPPD), mannitol, cyanidanol; and    vitamin E, and/or-   2) dichloroacetic acid to reduce the lactate which is formed during    the preservation.

The amount of antioxidants and/or dichloroacetic acid to be used is wellknown to the expert of the art, and widely reported in literature.

The solution according to the present invention is suitable to be usedto prevent the mechanisms which cause damage to organs and therefore isa solution which (a) prevents or reduces intracellular acidosis; (b)prevents the damage caused by oxygen free radicals, in particular duringreperfusion; (c) allows for the regeneration of high energy phosphatesduring reperfusion; (d) protects from expansion of the intracellularspaces; (e) sustains cellular metabolic requests.

The protective activity of the solution according to the invention hasbeen evaluated in suitable experimental models, having as referencesknown solutions, useful for the same purpose.

The following examples further illustrate the invention.

EXAMPLE 1

Many studies have demonstrated a correlation between the capacity of thetransplant organ to regenerate high energy phosphorylated compounds,such as ATP, and the success of the transplantation. In fact, one of themost important aspects, for example in the storage of liver, iscertainly the capacity for regeneration of high energy phosphorylatedcompounds, such as ATP, after the transplant.

The high-energy phosphorylated compounds must be available for a largenumber of regulatory mechanisms which prevent cellular damage. Forexample, the levels of NTP (nucleoside triphosphates) in the liver mustbe in sufficient quantity to restore critical cellular processes, suchas the maintenance of gradients which regulate the ion exchange acrossthe plasma and mitochondrial membranes, protein synthesis, bileproduction and the urea cycle.

In the experimental trials carried out to demonstrate the efficacy ofthe solution according to the present invention, isolated rat liverswere used, in which the alterations in ATP, ADP and total adeninicnucleotides (NTP) levels were analysed, through spectroscopy ³¹P-NMR (J.Lab. Clin. Med. 1992, 120: 559; Transplantation 1994, 57: 1576), duringcold ischemia, during the period of conservation, and during thereperfusion.

The realisation of these experimental trials required the resolution ofthe following principal problems:

-   1) the construction of a perfusion chamber adapted to work inside an    NMR magnet, and the creation of the pertinent perfusion circuit;-   2) the construction of a radio frequency (RF) coil utilisable with    the perfusion chamber;-   3) the definition of times (duration of the perfusion, conservation    and reperfusion phases; intervals to study through spectroscopy    during each of these phases; minimum duration time of a single    spectrum; optimisation of the passage times from one phase to    another).

Perfusion Circuit and Chamber

The diagram of the perfusion circuit is reported in FIG. 1.

In FIG. 1A the perfusion circuit includes a container maintained at 37°C. and supplied with a mixture of oxygen (95%) and carbon dioxide (5%),peristaltic pumps and sampling ports. FIG. 1B within the NMR magnet isthe oxygenation and thermostatisation apparatus includes a heat/oxygenexchanger 4 and a perfusion device maintained at 37° C. with a flux of25 ml/min.

The construction of this circuit was carried out taking into accountthat the appropriate temperature and oxygenation conditions must bemaintained in the organ, which is located at a distance of severalmeters from the perfusion system, constituted of pumps and a thermostat.In fact, these instruments need to be positioned outside the magneticfield of the measuring instruments, corresponding to a minimum distanceof 6 meters, in our case.

The perfusion medium KH (Krebs-Henseleit+glucose+BSA) (400 ml) (Krebs H.A. 1930; Biochem. J. 98, 720; Henseleit K. 1932; Hoppe-Seylerr's Z.physiol. Chem. 210, 33.), placed in a thermostatic container, away fromthe magnets, was continuously mixed by magnetic stirring.

The thermostatisation was performed in so as to obtain a temperature of37±0.5° C. in the perfusion chamber containing the organ inside themagnets.

Oxygenation of the medium was ensured through the use of a membraneoxygenator.

The oxygen saturation level was calculated on the basis of thepermeability factor of the silicone used, the calibre and thickness ofthe tube, the flow rate and the length of the tube wrapped in a spiralinside the oxygenation and thermostatisation apparatus. The valuescalculated gave results between 30 and 35% O₂.

The perfusate was recycled with the aid of a peristaltic pump with a 25ml/min flow rate.

The perfusion circuit used polyethylene tubes having a 1 mm innerdiameter of and a length of 6 meters to and from the magnets.

To ensure the maintenance of a controlled temperature inside theperfusion chamber in the magnet, both the thermostatisation andperfusion circuits were made to pass jointly inside a thermo isolatedneoprene tube.

The circuit was activated and stabilised at least one hour prior to theorgan perfusion to ensure reaching the desired temperature andoxygenation.

The perfusion chamber, constructed in Perspex, was fixed to a PVCsupport to allow insertion inside the magnets. That had a cylindricalgeometry, a diameter of 34 mm×65 mm in height.

The liver was maintained suspended at the desired height by fixing thecannula with appropriate distancing support disks. Drainage wasperformed by collecting the perfusion liquid in the funnel-shapedbottom, followed by aspiration to the collecting reservoir.

Normothermic damage by ischemia and reperfusion is a determining factorin the pathogenesis of hepatic damage, that arises during surgicalprocedures, such as hepatic resection and liver transplant.

In order to minimise as much as possible the heat ischemia time,variations to the standard surgical procedure of liver removal wereintroduced. In particular, non fasted animals were used; in animalmodels, in fact, studies in vivo and in vitro have demonstrated thatfasting aggravates the normothermic ischemic damage caused by areduction in glycogen content.

Male Wistar rats were used with initial body weights of 150 g. Theanimals were anaesthetised by an intraperitoneal injection (i.p.) ofsodium thiopental, then subjected to a median incision with successiveopening of the peritoneum.

The portal vein and the vena cava inferior were exposed, as muchadherences and fat as possible were removed to make removal of the organfaster and minimise the time of ischemia.

Ligatures were prepared for the vena cava inferior and the portal vein.

The vena cava was closed followed by, in rapid succession, the portalvein.

A cannula was inserted into the portal vein (Abbocath-T 20G; Abbott)which was fixed with a previously prepared suture and connected to asyringe containing cold Ringers lactate solution (Dawson R. M. C. (ed.),Elliott D. C. Elliott W. H. and Jones K. M. (1969) Data for biochemicalresearch, 2^(nd) ed. Clarendon Press, Oxford.) for a first blood washingperfusion. The vena cava was cut to allow the outflow of perfusionliquid.

Then the organ was removed as quickly as possible.

From closing the blood vessels to beginning the perfusion inside themagnets was a time interval no greater than 10 minutes, with 1-2 minutesof heat ischemia.

The cannula was left in situ and used for the reperfusion.

First the organ was removed.

Livers were perfused in situ with Ringers lactate solution at 4° C. toeliminate the blood and to limit as much as possible the normothermicischemia times of the organ.

The isolated organs were then placed in the perfusion chamber inside themagnets, in an appropriate bioreactor, fed with Krebs-Henseleit solutionat 37° C.±0.5° C., 35% 35% O₂-5% CO₂, then the basal reference spectra³¹P NMR (time 0) were acquired.

The acquisition of these NMR spectra was carried out to eliminate thebiological variability between organs; in this manner, in fact, thevariations observed after preservation can be referred to valuesdetermined in the same organ immediately upon removal from the animaland stabilised in perfusion for 40′.

At the end of 40′ stabilisation, the organs were perfused with differentstorage solutions (Carnival or UW) at 4° C.

The experimental tests were carried out using Carnival solution inaccordance with the present invention, and two known storage solutionswere used as reference:

-   1) UW solution with the addition of insulin (40 I.U./L) and    dexamethasone (8 mg/L); and-   2) EuroCollins solution.

Table 4 reports the compositions of Carnival, EuroCollins and UWsolutions.

TABLE 4 Carnival EuroCollins UW solution solution solution K₂HPO₄•3H₂O32 mM 32 mM 2.5 mM  KH₂PO₄ 15 mM 15 mM − KCl 15 mM 15 mM − NaHCO₃ 10 mM10 mM − Glucose 187.5 mM   194 mM  − Isovaleryl L-carnitine  2 mM − −Fumarate L-carnitine (inner salt) 10 mM − − Glycine − − 15 mMAllopurinol − −  1 mM Adenosine − − 2.5 mM  Lactobionate − − 100 mM MgSO₄•7H₂O − −  5 mM Raffinose − − 30 mM PEG\ − − + “−” means “absent”;“+” means “present”.

In some experiments spectra were acquired cold, immediately afterperfusion of the organ with the solution in accordance with the presentinvention, to evaluate the kinetics of the disappearance ofphosphorylated metabolites in the 1^(st) hour.

The results obtained are reported in tables 8-10.

For each spectrum it was noted:

-   1) the total levels of phosphorylated metabolites;-   2) the levels of inorganic phosphate and phospho monoesters    (P_(i)+PME);-   3) the levels of nucleoside triphosphates (NTP);-   4) the levels of NAD as the sum of the signals α-NTP+NAD.

The spectra were acquired at 10-minute intervals over one hour.

The livers were stored by immersion in several solutions at 4° C. for atotal time of 26 hours.

The storage time was chosen in order to evaluate the restarting capacityafter a much longer time than that normally used in clinical practice orin experiments.

At the end of the storage time (26 hours), the livers were again placedinside the magnet in perfusion with solution KH, at 37° C., 35% O₂, tomonitor the phosphorylated metabolites.

³¹P-NMR spectra were acquired every 15′ over a time limit of 140′.

The results obtained are reported in tables 5-7.

NMR Experimental Conditions

The specific characteristics of the ³¹P-NMR experiment strictly dependedon the experimental configuration chosen for liver perfusion, with themain objective of obtaining spectra having optimal signal/noise ratios.

The salient difficulties to take into consideration were:

-   i) the necessity to use brief scanning times;-   ii) optimisation of the operating conditions of the Nuclear Magnetic    Resonance system;-   iii) heterogeneity in the geometry and composition of the samples.

The experimental conditions utilised were:

-   -   spectral bandwidth 8.33 KHz;    -   sampling 2048 points;    -   acquisitions 900;    -   dummy scan 2;    -   2-step phase cycle;    -   impulse at 90° (150 μs);    -   2 s repetition interval.

The spectra, in relation to the diverse experimental conditions wereobtained from the accumulation of 450 scans in conditions of perfusionor reperfusion and of 300 scans in conditions of hypothermia, alwayswith a repetition time of 2 seconds.

The ³¹P-NMR spectra obtained display signals relative to α, β and γphosphate of the nucleotide triphosphates respectively at −9.7, −18.35and −4.2 ppm and are prevalently represented by adenosine triphosphate(ATP) and in minor amounts by guanosine triphosphate (GTP), uridinetriphosphate (UTP) e cytidine triphosphate (CTP).

The signal assigned to αNTP is contributed to by the resonance of thenicotinamide-adenine-dinucleotide phosphates (NAD) andnicotinamide-adenine-dinucleotide phosphate.

The signals relating to α and β phosphates of the nucleotidediphosphates, mainly represented by adenosine diphosphate (ADP), arerespectively at −8.9 and −4.4 ppm. This last signal is partially maskedby the signal from γNTP.

The signals at −11.5 ppm attributed to compounds having twodi-esterified phosphate groups (DPDE) are mostly represented by uridinediphosphoglucose and uridine diphospho glucuronate.

Signals relating to phospholipid intermediates are present in thespectral zone between 5.8 and 4 ppm, where the nuclei of phosphategroups from monophosphate esters (PME) resonate.

300 mM methylene diphosphonate (MDP) contained in a capillary (0.5 ml)fixed to the inside of the perfusion chamber, was used as a reference.

The signal area was evaluated by applying one of the known programs forthe reconstruction of resonance spectra (program SPEC ANA; SMIS).

Results Obtained

The initial perfusion with KH solution (37° C., 35% O₂) after removaland washing of the liver with Ringers lactate solution (4° C.) for 40′,was deemed sufficient for the stabilisation of phosphorylated metabolitelevels.

The initial levels (pre-storage) of β-ATP resulted as being 8.3±3.1,these for α-ATP of 22.6±5.4 respectively (whilst the values reported inthe table are expressed as a percentage of the reference).

The ratios β-ATP/Pi+PME, α-ATP/Pi+PME and β-ATP/α-ATP gave resultsrespectively of 8.4±5 , 23.4±10.2 and 35.7±11.

The values thus obtained were used as reference for time 0 for thesubsequent evaluations.

In Table 5 the results relating to a representative spectrum obtainedpreviously, during and after preservation with Carnival solution arereported.

TABLE 5 TIMES β γ α Stabilisation of levels; (NMR analysis afterexplant, washing, perfusion with KH, at 37° C.) 40′ 8.9 10.3 27.5Perfusion with Carnival solution and storage at 4° C.; NMR analyses wereperformed at 4° C. at the times indicated below 10′ n.d. n.d. 58% 20′n.d. n.d. 74% 30′ n.d. n.d. 69% 40′ n.d. n.d. 65% 50′ n.d. n.d. 67% 60′n.d. n.d. 71% The organs were conserved immersed in Carnival solution,at 4° C., for further 25 hours The organs were then perfused at 37° C.with KH solution; NMR analyses were performed at 37° C. at the timesindicated below 30′ 84% 68% 90% 45′ 81% 81% 87% 60′ 109%  75% 99% 75′112%  97% 83% 90′ 71% 83% 70% 105′  99% 85% 87% 120′  101%  68% 68%135′  72% 69% 75%

The values relating to after explant are expressed as ratios of thereference signal. All other values are expressed as percentages withrespect to the latter.

In Table 6, data relating to a previously obtained representativespectrum, during and after storage with UW solution, are reported.

TABLE 6 TIMES β γ α Stabilisation of levels; (NMR analyses afterexplant, washing, perfusion with KH, at 37° C.) 40′ 5.8 4.8 21.8Perfusion with UW solution and storage at 4° C.; NMR analyses werepreformed at 4° C. at the times indicated below 10′ 51% 71% 117%  20′28% 56% 96% 30′ n.d. 64% 95% 40′ n.d. 80% 128%  50′ n.d. 44% 75% 60′n.d. n.d. 81% The organs were stored immersed in UW solution, at 4° C.,for a further 25 hours The organs were then perfused at 37° C. with KHsolution; NMR analyses were performed at 37° C. for the times indicatedbelow 30′ 51% 49% 79% 45′ 45% 56% 70% 60′ 91% 74% 90% 75′ 79% 62% 81%90′ 65% 94% 75% 105′  58% 58% 86% 120′  56% 91% 83% 135′  44% 73% 64%

Values relating to after explant are expressed as a ratio of thereference signal. All other values are expressed as a percentage of thelatter.

In table 7, data relating to a previously obtained representativespectrum, during and after storage with EuroCollins solution, arereported.

TABLE 7 TIMES β γ α Stabilisation of levels; (NMR analyses afterexplant, washing, perfusion with KH, at 37° C.) 40′ 7.9 6.1 25.6Perfusion with EuroCollins solution and storage at 4° C.; NMR analyseswere performed at 4° C. at the times indicated below 10′ n.d. n.d. 96%20′ n.d. n.d. 69% 30′ n.d. n.d. 67% 40′ n.d. n.d. 58% 50′ n.d. n.d. 65%60′ n.d. n.d. 51% The organs were stored immersed in EuroCollinssolution, at 4° C., for a further 25 hours The organs were then perfusedat 37° C. with KH solution; NMR analyses were performed at 37° C. at thetimes indicated below 30′ 0% 0%  0%

The values relating to after explant are expressed as ratios of thereference signal. All other values are expressed as percentages of thelatter.

In table 7, data from a single reperfusion (30′) spectrum are reportedsince at this and subsequent times, no re-synthesis of phosphorylatedcompounds was observed.

The kinetics of the disappearance of phosphorylated metabolites observedin the first 60′ of cold preservation has demonstrated that the signalof α-ATP+α-ADP+NAD is present until the end of the measurements for allthe preservation solutions in a manner that varies from organ to organ.

The β-ATP signal was detectable up to a maximum time of 30′ only withpreservation with UW, whilst it was absent even at the first spectrumfor EuroCollins and Carnival solutions.

The γ-ATP+β-ADP signals remained present up to 50′ of acquisition, duehowever solely to the presence of ADP.

The residual quantity of phosphorylated metabolites at 60′ did notcorrelate with the capacity for re-synthesis of ATP in the normothermicreperfusion after 26 hours of conservation in the different solutions.

In tables 8, 9 and 10 values with standard deviations for β-ATP andα-ATP acquired at 80′ and 140′ of normothermic perfusion after perfusionand at the beginning of the preservation at 4° C. (cold spectra, NMRanalyses were performed about 2 hours after organ removal) in Carnival,UW and EuroCollins solutions respectively, are reported.

TABLE 8 Time β-ATP α-ATP Solution (min) (n = 8) (n = 8) Carnival  80′62.4 ± 24   62.4 ± 14   Carnival 140′ 55.2 ± 25.2 53.5 ± 15.6 (n is thenumber of animals used)

TABLE 9 Time β-ATP α-ATP Solution (min) (n = 8) (n = 8) UW  80′ 64.2 ±13.1 55.2 ± 17 UW′ 140′ 57.2 ± 13.5 63.4 ± 19 (n is the number ofanimals used)

TABLE 10 Time β-ATP α-ATP Solution (min) (n = 8) (n = 8) Euro Collins 80′ 15 ± 13.7   52 ± 14.4 EuroCollins 140′ N.D. 25.4 ± 21 (n is thenumber of animals used)

The values are expressed as percentages with reference to the samesignal acquired at 37° C. after explant.

As can be noted, the solution according to the present invention plays aprotective role in cellular vitality during the phase of normothermicreperfusion of isolated liver permitting the re-synthesis of ATP whichis comparable during the phase of normothermic reperfusion to thatobserved with UW. It is important to remark that the reappearance ofsignal, and therefore the capacity for re-synthesis, is faster for theorgans stored in Carnival with respect to these stored in UW, wheresatisfactory re-synthesis is not observed up to 60′.

Vice versa, organs stored in EuroCollins solution do not demonstrateappreciable ATP levels in the times considered.

Furthermore, it should be underlined that the concentrations of themetabolites were maintained at comparable levels for UW and Carnivalsolutions up to 140′.

The solution according to the present invention, in the hypothermicstorage phase, further prevents cellular swelling due to osmoticphenomena, as observed qualitatively, analogous to that obtained with UWsolution with lactobionate, raffinose and glycine added.

The solution according to the present invention favours the maintenanceof hepatic bioenergetic integrity even after 26 hours of hypothermicconservation providing, at lower cost, comparable results to theseobtained with the more expensive UW.

1. A solution comprising: a) K₂PO₄•3H₂O 10-60 mM b) KH₂PO₄ 3-50 mM c)KCl 3-50 mM d) NaHCO₃ 2-50 mM e) glucose 50-250 mM f) isovalerylL-carnitine 2 mM g) L-carnitine inner salt 2 mM

wherein the solution is used to maintain a perfused organ awaitingtransplantation, wherein the organ is selected from the group consistingof heart, liver, pancreas, lung and kidney.
 2. The solution according toclaim 1, wherein the organ to stored is liver.
 3. The solution accordingto claim 1, further containing at least one anti-oxidant in an amountsufficient to inhibit the formation of oxygen derived free radicals. 4.The solution according to claim 3, wherein the anti-oxidant is selectedfrom the group consisting of allopurinol, glutathione, beta-carotene,catalase, superoxide dismutase, dimethyl thiourea (DMTU), diphenylphenylene diamine (DPPD), mannitol and cyanidanol.